The polymerase chain reaction (PCR) is the most widely used method for in vitro DNA amplification for purposes of molecular biology and biomedical research. This process involves the separation of the double-stranded DNA in high heat into single strands (the denaturation step, typically achieved at 95-97° C.), annealing of the primers to the single stranded DNA (the annealing step) and copying the single strands to create new double-stranded DNA. PCR is commonly carried out in bench-top machines that are large, expensive, costly to run and maintain. This can limit the potential applications of DNA amplification in situations outside the laboratory (e.g., in the identification of potentially hazardous micro-organisms at the scene of investigation or at the point of care of a patient). In vivo, DNA is replicated by DNA polymerases with various accessory proteins, including a DNA helicase that acts to separate the DNA by unwinding the DNA double helix. Helicase-dependent-amplification (HDA) was developed using a helicase (an enzyme) to denature the DNA.
In HDA, strands of double stranded DNA are first separated by a DNA helicase and coated upon by single stranded DNA (ssDNA)-binding proteins. In the second step, two sequence specific primers hybridize to each border of the DNA template. DNA polymerases are then used to extend the primers annealed to the templates to produce a double stranded DNA. The two newly synthesized DNA products are then used as substrates by DNA helicases, entering the next round of the reaction. Thus, a simultaneous chain reaction develops, resulting in exponential amplification of the selected target sequence.
As opposed to PCR, the HDA process takes place at a constant (isothermic) incubation temperature and does not require a bench-top thermocycler. However, as the enzyme helicase facilitates strand separation, it is limiting during reaction kinetics. The helicase used for HDA is of the UvrD type of E. coli. Homologue thermostabile helicases may also be used (tHDA).
During HDA, the substrate of the helicase is the double stranded amplicon which is generated during the amplification reaction. Because the helicase cannot facilitate strand separation from the middle of a dsDNA fragment, the structure of the ends of the amplicon are very important. The ends of an amplicon are defined by the primers designed for a given template. Consequently, primer design is crucial for helicase kinetics and overall HDA reaction success.
U.S. application Ser. No. 10/665,633 describes factors to be considered in HDA primer design. Generally, primer pairs suitable for use in HDA are short synthetic oligonucleotides, for example, having a length of more than 10 nucleotides and less than 50 nucleotides. Oligonucleotide primer design involves various parameters such as string-based alignment scores, melting temperature, primer length and GC content (Kampke et al., Bioinformatics 17:214 225 (2003)). When designing a primer, one of the important factors is to choose a sequence within the target fragment which is specific to the nucleic acid molecule to be amplified. The other important factor is to decide the melting temperature of a primer for HDA reaction. The melting temperature of a primer is determined by the length and GC content of that oligonucleotide. Preferably the melting temperature of a primer is about 10 to 30° C. higher than the temperature at which the hybridization and amplification will take place. For example, if the temperature of the hybridization and amplification is set at 37° C. when using the E. coli UvrD helicase preparation, the melting temperature of a pair of primers designed for this reaction should be in a range between about 47° C. to 67° C. If the temperature of the hybridization and amplification is 60° C., the melting temperature of a pair of primers designed for that reaction should be in a range between 65° C. and 90° C. To choose the best primer for a HDA reaction, a set of primers with various melting temperatures can be tested in parallel assays. More information regarding primer design is described by Kampke et al., Bioinformatics 17:214 225 (2003).
Hairpin structures within primers may reduce primer binding kinetics which in turn, can affect efficiency and speed of the amplification process. Self-complementarity of primers can result in nonspecific primer-dimer amplicons that compete with the amplification of the specific target amplification. As such, primer specificity, melting characteristics, hairpin structures, self-complementarity must all be taken into account when designing HDA primers.
There is a need in the art for better HDA primers and methods of their use and design.